Chiral integrin modulators and methods of use thereof

ABSTRACT

The invention provides enantiomers of ajoene and derivatives of Z(−)-ajoene. The derivative of Z(−)-ajoene are useful for modulating integrin-mediated functions, for treating disorders, diseases or conditions in which integrins play a role, and for treating tissues to improve their condition for a subsequent use, such as transplantation.

RELATED APPLICATIONS

This application is a continuation of prior application Ser. No.10/332,545, filed Jun. 17, 2003, which is the National Stage ofInternational Application No. PCT/US01/21826, filed Jul. 10, 2001, whichclaims the benefit of U.S. Provisional Application No. 60/217,651, filedJul. 10, 2000. The entire contents of all three of the above-identifieddocuments are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Integrins are heterodimeric transmembrane glycoproteins which, interalia, act as cell receptors for various entities, herein termedcollectively “integrin ligands,” including, for example, surfacemolecules of other cells and extracellular matrix (ECM) proteins. Bothsoluble and immobilized integrin ligands are known to be ordinarilybound by integrins. Integrins are found on most types of cells. Ligandbinding by integrins may result in occur in association with a series ofadditional cellular events involving one or more cellular functions.These cellular events and functions, some of which are discussed belowfor illustrative purposes, are termed “integrin-mediated.” For a generalreview of integrins, see, Guidebook to the Extracellular Matrix andAdhesion Proteins (Kreis, et al., Eds.), 1993, and Pigot, et al., TheAdhesion Molecule Facts Book, Academic Press, 1993.

One such integrin-mediated cellular function is signaling. For instance,certain integrins are known to transfer information from the inside tothe outside of the cell (inside-out signaling) or from the outside tothe inside of the cell (outside-in signaling), although other types ofsignaling may also occur, as may combinations thereof. An exampleinvolving inside-out signaling is the process whereby an integrinacquires or expresses affinity for ligands in response to intracellularevents (integrin upregulation). Binding of integrin ligands to certainintegrins (e.g., in the case of integrin-mediated cell adhesion) mayinitiate signal transduction events, in a manner similar to thatdescribed for other cell surface receptors. Signals thus elicited aretermed outside-in signals and are involved in the regulation of variouscell responses, which may include gene expression, cell differentiation,and cell proliferation.

Signaling may result in the clustering of cellular molecules inlocalized areas of cellular membrane, e.g., in the association ofintegrins with each other (and other molecules) by lateral interactions.The formation of such clusters may influence various integrin functionsin multiple ways, including, for example, by additional or secondarysignaling events or interactions, and by altered ligand affinity.

The integrin-mediated function of adhesion is, or variousintegrin-mediated events associated with adhesion are, important for avariety of physiological and pathological responses. The extent ofadhesion is functionally related to integrin signaling. For example, inassociation with initial integrin-dependent adhesion to a substratum,certain cells change their shape and start spreading on the surface ofthe stratum, using integrins for establishing new contacts with theunderlying proteins (e.g. extracellular matrix (ECM)components). Inmotile cells, the whole array of integrin-mediated events involvingadhesion—initial contact, cell shape change, cell spreading, and celllocomotion—is sometimes termed “the adhesion cascade” (Sharar, S. R, etal., The Adhesion Cascade and Anti-Adhesion Therapy: An Overview, 16Springer Semin. Immunopathol. 359, 1995). Adhesion cascades are viewedas integral to one or more familiar cell motility patterns, includingangiogenesis, lymphocyte homing, tumor cell metastasis, and cellmigration processes associated with wound healing, although similarcascade mechanisms are also viewed as operative even in the absence ofcell locomotion (e.g., in platelet adhesion and aggregation).Extravasation of neutrophils is described below in greater detail, as aparadigmatic integrin-mediated adhesion cascade (Hub, E., et al.,Mechanism of Chemokine-Induced Leukocyte Adhesion and Emigration,Chemoattractant Ligands and Their Receptors (Horuk, R., Ed.), BocaRaton, CRC Press, 1996, 301).

The onset of extravasation is heralded by the appearance in thecirculation of chemotactic factors, or chemoattractants (i.e., specificsubstances that initiate cell migration along their concentrationgradients). Chemoattractants (e.g., chemokines, bacterial peptides, andproducts of complement activation) activate neutrophils to upregulatetheir integrin receptors (neutrophil integrins include, e.g., LFA-1[CD11a/CD18], CR3 [also known as Mac-1, CD11b/CD18], and gp150,95[CD11c/CD18]). Neutrophils thus activated adhere to endotheliocytes,change shape, and spread on the endothelial surface. Thereafter, thestimulated motile apparatus of the neutrophils gives rise to migration,and the neutrophils start moving, first across the endothelial layer andfurther, through the perivascular ECM, towards the source of thechemotactic stimulus, e.g., pathogenic bacteria invading a certainbodily tissue. During the whole process, from the initial fin contactwith the endothelium to the cessation of locomotion at the destinationsite, various integrins serve to attach the neutrophil to the substratait encounters, enabling its recruitment to the locus of infection.

Another integrin-mediated function is cell-cell fusion. Underphysiological conditions, fusion is a developmentally regulated stage inthe differentiation of certain multinucleate cells (e.g., osteoclasts,myocytes, and syncytiotrophoblasts), and fusion is also a prerequisiteto fertilization (in the case of sperm-egg fusion). Fusion is effectedby specialized cellular systems involving integrins (see, e.g., refs.cited in Huovila, A.-P. J., et al., ADAMs and Cell Fusion, 8 CurrentOpin. Cell. Biol. 692, 1996 and Ohgimoto, S., et al., Molecular:Characterization of Fusion Regulatory Protein-1 [FRP-1] that InducesMultinucleate Giant Cell Formation of Monocytes and HIV gp160-MediatedCell Fusion: FRP-1 and 4F2/CD98 Are Identical Molecules, 155 J. Immunol.3585, 1995).

The ability to undergo recirculation from intracellular compartments tothe cell surface and vice versa is a common property of divers cellularreceptors, including integrins (see, e.g., Handagama, P., et al.,Kistrin, an Integrin Antagonist, Blocks Endocytosis of Fibrinogen intoGuinea-Pig Megakaryocyte and Platelet alpha-Granules, 91 J. Clin.Invest. 193, 1993). This capability of integrins facilitates themediation of other cellular functions by transporting into the cellextracellular material (e.g., soluble proteins, particulate matter, andother cells). Integrin-mediated internalization is used by certainmicroorganisms to invade their targets. For example, CR3 mediates entryof iC3b-opsonized HIV-1 and HIV-2 into CD4-negative lymphocytic andmonocytic cells Ooyer, V., et al., Complement Mediates Humaninmunodeficiency Virus Type I Infection of a Human T cell Line in a CDSand Antibody-Independent Fashion, 173 J. Exp. Med. 1151, 1991).

The above-delineated functions of integrins are illustrative only, asother characterizations of integrin functions can also be made.Moreover, the integrin-mediated functions as delineated herein areoverlapping and interrelated. In the case of neutrophil extravasation,for example, the initial chemotactic signal activating the cells iscommonly functionally associated with in integrin upregulation(inside-out signaling) and adhesion to the endothelial surface. Thisadhesion event, in turn, is associated with outside-in signal, enablingthe neutrophil to change its shape, which is a prerequisite to thespreading and migration of the cell. Likewise, when the neutrophil thathas arrived to the source of chemoattractants establishes an adhesiveinteraction with the bacteria by means of integrins, an outside-insignal is transduced, which is associated with the initiation ofinternalization of the integrins involved, together with the bacteriaattached thereto (phagocytosis).

Furthermore, regarding outside-in integrin signaling, certain cellularprocesses are co-mediated by several signaling systems acting inconcert. In the case of neutrophils extravasating to the tissues tophagocytose bacteria, the neutrophils receive signals by means of thereceptors of the chemoattractant (along the concentration gradient ofwhich the movement occurs) and by means of distinct integrins (thosethat attach it to the substratum and, subsequently, those recognizingthe bacteria). This interplay of signals mediates the antibacterialmachinery of the neutrophils with the consequence that only upon contactwith the bacteria, which is established by means of a particular type ofintegrin, are the constituents of the intracellular granules releasedand reactive oxygen species formed. As a result, the formation andrelease of microbicidal substances take place preferentially at sites ofcontact with bacteria, enabling effective killing of the bacteria andpreventing the destruction of host tissue (Wright, S. D., Receptors forComplement and the Biology of Phagocytosis [Chapter 25], Inflammation:Basic Principles and Clinical Correlates [Gallin, J. I., et al., Eds.],2nd Ed., New York, Raven Press, 477, 1992).

Clearly, a broad range of cellular activities can be regulated bymodulating certain integrin functions with appropriate agents. One suchintegrin-modulating agent is ajoene(4,5,9-trithiadodeca-1,6,11-triene-9-oxide):

Ajoene, and a precursor thereof, can be isolated from products derivedfrom extracts of garlic (Allium sativum). As the garlic is crushed,alliin in the garlic comes into contact with alliinase in the cell wallto form allicin. Then, in the presence of a polar solvent, allicin mayform ajoene.

Ajoene has been previously shown to inhibit platelet aggregation byinactivating allosterically the platelet integrin, GP IIb/IIIa(Apitz-Castro, R., et al., 141 Biophys. Res. Commun. 145, 1986). It hasbeen demonstrated that stereoisomers of ajoene (i.e., E- andZ-4,5,9-trithiadodeca-1,6,11-triene-9-oxides) exhibit no significantdifferences in their effects on platelets (Block, E., et al., 108 J. Am.Chem. Soc. 7045, 1986). For this reason, most of subsequent studies ofthe integrin modulation by ajoene were carried out on various mixturesof the E- and Z-isomers. It was shown, for example, that ajoene is apotent inhibitor of a wide variety of adhesion-dependent processes,including neutrophil aggregation, HIV transmission (Tatarintsev, A. V.,et al., 6 AIDS 1215, 1992), and tumor metastasis. U.S. Patents issued toTatarintsev, et al disclose the use of ajoene for treatment ofinflammation (U.S. Pat. No. 5,948,821), arthritis (U.S. Pat. No.5,856,363), and tumors (U.S. Pat. No. 5,932,621), as well as forcontraception (U.S. Pat. No. 5,863,954) and inhibition of immuneresponses (U.S. Pat. No. 5,863,955). All of these diseases andconditions involve integrin-mediated processes. See also PCT applicationWO 97/25031, which describes the use of ajoene to treat additionaldiseases and conditions which involve integrin-mediated processes.

The presence of the sulfoxide group in the molecule of ajoene (aprerequisite to optical isomerism) and stereoisomerism of the compound(around the double bond at carbon 6) create a possibility for fouroptical isomers (enantiomers): E(R)-, E(S)-, Z(R)-, andZ(S)-4,5,9-trithiadodeca-1,6,11-triene-9-oxides. This possibility hasnever been suggested in the art. Moreover, in the case of allicin (whichalso contains a sulfoxide radical), even the existence of opticalactivity has been questioned, so that the existence of enantiomers, letalone stable enantiomers, would have been considered unlikely (Garlic:The Science and Therapeutic Application of Allium Sativum L. and RelatedSpecies, page 56 (Lawson L. D., et al., Eds., 1997)).

OBJECTS OF THE INVENTION

An object of the present invention is to provide enantiomers of ajoene.

Another object of the present invention is to characterize theintegrin-modulating activity of the enantiomers, with a view toidentifying species that would differ in activity from racemic mixtures.The word “racemic” is used herein in its strict sense to designate achemical compound that contains equal quantities of dextrorotatory andlevorotatory enantiomers and, therefore, does not rotate the plane ofincident polarized light (The American Heritage Dictionary of theEnglish Language, 1996).

Yet another object of the present invention is to provide methods ofpreparing ajoene enantiomers.

A final object of the present invention is to provide stereoselective,chiral integrin modulators and methods of use thereof.

SUMMARY OF THE INVENTION

The objects of the invention have been attained. The invention providesenantiomers of ajoene: E(+)-, E(−)-, Z(+),- andZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxides. The invention furtherprovides methods of preparing the enantiomers. The invention alsoprovides approaches to, and methods of, assessment of theintegrin-modulating activity of the enantiomers. The invention furtherprovides stereoselective, chiral integrin modulators (CIMs), as furtherdefined and specified herein. Specifically, such modulators includederivatives of Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide of theformula:

wherein:

each X₁ may be the same or different than the other X₁ and each X₁ ishydrogen, an alkyl, an alkenyl, an alkynyl, an aryl, an aryl substitutedwith one or more —NO₂ groups, an aryl substituted with one or more loweralkyls, a group of the formula RO—, a group of the formula RCO—, a groupof the formula RCOO—, a group of the formula ROCO—, a group of theformula (R)₂N—, a group of the formula RCON—, a group of the formulaRN═N—, a group of the formula RS—, a group of the formula RSO₂—, a groupof the formula RSO—, a group of the formula RSO₂O—, a group of theformula RSOO—, a halogen atom, ammonio, nitrozo, nitro, mercapto, orsulfo;

R is hydrogen, lower alkyl, aryl, or an aryl substituted with one ormore lower alkyls;

m is 0-30; and

n is 0-30.

The prototypal CIM of this invention isZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.

The invention also provides methods of using the above CIMs. First, theinvention provides a method of modulating an integrin-mediated functionof one or more cells using the CIMs. Second, the invention providesmethod for the treatment of a variety of disorders, diseases andconditions. In particular, the invention provides: (1) methods oftreating or preventing a disorder, disease or condition in which one ormore integrins play a role; (2) methods of treating or preventingthrombotic disorders and diseases/conditions arising therefrom(embolism, ischemia, infarction, etc.); (3) methods of treating orpreventing inflammation and inflammatory diseases; (4) methods oftreating, preventing, or inhibiting the transmission of viralinfections; (5) methods of treating shock; (6) a method of treatingarthritis; (7) methods of contraception; (8) methods of treating orsuppressing adverse, undesirable or self-destructive immune responses,including acute and chronic hypersensitivity reactions (such asanaphylaxis and allergy), transplant rejection, and graft-versus-hostdisease (GVHD); (9) methods of treating autoimmune diseases; (10)methods of inhibiting undesirable integrin-mediated cell-cell fusion;(11) methods of inhibiting the formation of lesions; (12) methods oftreating psoriasis; (13) methods of treating atherosclerosis; (14)methods of treating diseases or conditions involving a plurality ofintegrin-dependent etiopathogenetic mechanisms; (15) methods ofinhibiting the transfer of genetic material; and (16) methods oftreating cancer, preventing metastasis of tumors, or inhibiting certain(integrin-mediated) types of carcinogenesis.

The invention further provides-pharmaceutical compositions. Thecompositions comprise the above CIMs and a pharmaceutically-acceptablecarrier.

In addition, the invention provides a method of treating a tissue bycontacting the tissue with a CIM. Such treatment improves the conditionof the tissue for subsequent use, as compared to tissue which is nottreated with a CIM. In particular, tissue which is to be transplantedinto a recipient maybe treated with a CIM prior to, in the course of,and/or after harvesting, and the chances of the tissue beingsuccessfully transplanted will be increased.

Finally, the invention provides a kit for treating tissue. The kitcomprises a container holding one or more of the above CIMs.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph of HIV-induced syncytium formation as a percentage ofuntreated control versus concentration (micromoles per liter) of theunseparated 3:1 mixture of racemic Z- and E-ajoenes (♦), Z(+)-ajoene(+), racemic E(−)ajoenes (◯), and Z(−)-ajoene (X) (curves 1 through 4,left to right, respectively).

For all the curves, the effect of ajoene enantiomers and enantiomermixtures on the fusion of cultured, intact H9 cells withHIV-1_(RF)-infected H9 cells is disclosed. The vertical graph axisexpresses the maximum amount of syncytia formed in the absence of thecompounds (100 percent), while the points on the curves representpercentages of such an amount of syncytia formed in the presence ofvarying concentrations of the compounds and compound mixtures(micromoles per liter).

The coincidence of curves 1 through 3 and the leftward shift of curve 4demonstrate that Z(−)-ajoene is at least four times more active than itsZ(+) counterpart, racemic E-ajoene, or the unseparated 3:1 mixture ofracemic Z- and E-ajoenes (the respective values of IC₁₀₀ are 12.5 and 50micromoles per liter).

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

This invention provides enantiomers of ajoene: E(+)-, E(−)-, Z(+),- andZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxides. By this invention,ajoene maybe separated into the two pairs of enantiomers. Specifically,both (E) and (Z) racemic forms may be used as a starting material forthe separation, although various (E)/(Z) mixtures may also be used. Inone preferred embodiment, the enantiomers are structurally identified byat least two characteristics, including mobility in one or more organicsolvent mixtures and optical activity, both detected at the same fixedtemperature. Correspondingly, the enantiomers are designated E(+)-,E(−)-, Z(+),- and Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxides.

In addition, the invention discloses methods whereby theintegrin-modulating activity of ajoene enantiomers may be assessed. Onesuch method involves VLA-4-mediated adhesion of enzyme-labeled PM1 cellsto VCAM-1-coated artificial substrata. Specifically, the cells wereexposed to isolated ajoene enantiomers (or the vehicle thereof) andallowed to adhere to immobilized VCAM-1. Thereafter, the adherent cellswere lyzed and, following addition of the substrate of the enzyme andincubation, the activity of the enzyme was measuredspectrophotometrically. The value of this parameter, characterizing thenumber of the adherent cells, is inversely proportional to theintegrin-modulating activity of the compound.

Another such method is based on the inhibition of HIV-mediated syncytiumformation, a phenomenon known to depend on the functional activity ofintegrins. The purified Z enantiomers, racemic E-ajoene, and theoriginal 3:1 mixture of racemic Z- and E-ajoenes were taken up in DMSOat a concentration of 10 mg/mL. Dilutions were made in RPMI 1640 mediumcontaining 10% fetal calf serum and 10 mM HEPES (cRPMW).HIV-1_(RF)-infected H9 cells and uninfected H9 cells were washed withcRPMI and resuspended in cRPMI at a density of 4×10⁶ per mL. Uninfectedcells (50 μL) were mixed with serial dilutions of the compounds (100 μL)and incubated at 37° C. for30 minutes before adding and mixing 50 μL ofinfected H9 cells. The plates were incubated at 37° C. for 6 to 15 hoursbefore scoring syncytium formation.

By these methods, Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide was atleast four times more active as an integrin modulator than otherenantiomers, racemic E-ajoene, or the unseparated 3:1 mixture of racemicE and Z-ajoenes; The unexpected superior activity of one out of the fourenantiomers correlates with the presence in the molecule of thefollowing trithia oxide structure:

It is, therefore, understood that chiral compounds containing the abovecore structure comprise a class of potent integrin modulators.

Specifically, the class of chiral integrin modulators (CIMs) includescompounds of the formula:

wherein:

each X₁ may be the same or different than the other X₁ and each X₁ ishydrogen, an alkyl, an alkenyl, an alkynyl, an aryl, an aryl substitutedwith one or more —NO₂ groups, an aryl substituted with one or more loweralkyls, a group of the formula RO—, a group of the formula RCO—, a groupof the formula RCOO—, a group of the formula ROCO—, a group of theformula (R)₂N—, a group of the formula RCON—, a group of the formulaRN═N—, a group of the formula RS—, a group of the formula RSO₂—, a groupof the formula RSO—, a group of the formula RSO₂O—, a group of theformula RSOO—, a halogen atom, ammonio, nitrozo, nitro, mercapto, orsulfo;

R is hydrogen, lower alkyl, aryl, or an aryl substituted with one ormore lower alkyls;

m is 0-30; and

n is 0-30.

“Alkyl” means a straight-chain or branched-chain alkyl containing 1-10carbon atoms or a cyclic alkyl containing 3-7 carbon atoms. “Loweralkyl” means a straight-chain or branched-chain alkyl containing 1-4carbon atoms. Both terms include all isomers. Preferably, the alkyl ismethyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, iso-pentyl, neo-pentyl, n-hexyl, or cyclohexyl.

“Alkenyl” means a straight-chain or branched-chain alkenyl containing2-10 carbon atoms and at least one double-bond. The term includes allisomers. Preferably, the alkenyl is vinyl, propenyl, or iso-propenyl.

“Alkynyl” means means a straight-chain or branched-chain alkynylcontaining 2-10 carbon atoms and at least one triple bond. The termincludes all isomers. Preferably, the alkynyl is ethynyl, 1-propynyl, or2-propynyl.

“Aryl” means a group containing at least one aromatic ring. Preferably,the aryl is phenyl. The aryl may be substituted with one or more —NO₂groups, in which case it is preferably m-nitrophenyl, p-nitrophenyl,o-nitrophenyl, 3,5-dinitrophenyl, or 2,4-dinitrophenyl. The aryl mayalso be substituted with one or more lower alkyls, in which case thepoint of attachment to the sulfur atoms in formula (2) or to an atom ofone of the substituent groups listed above (e.g., RO—, RCO—, etc.) canbe by means of an alkyl group or a ring carbon. Preferred arylssubstituted with one or more lower alkyls are benzyl, o-tolyl, p-tolyl,m-tolyl, 3,5-xylyl, and 2,6-xylyl.

“Halogen” means fluoro, chloro, bromo, or iodo.

Preferred groups of the formula RO— are hydroxy, methoxy, ethoxy,phenoxy and benzyloxy.

Preferred groups of the formula RCO— are acetyl, formyl, and benzoyl.

Preferred groups of the formula RCOO— are formlyoxy or acetoxy.

Preferred groups of the formula ROCO— are methoxycarbonyl,ethoxycarbonyl, tert-butoxycarbonyl, and benzyloxycarbonyl.

Preferred groups of the formula (R)₂N— are amino, methylamino,ethylamino, phenylamino, dimethylamino, and diethylamino.

Preferred groups of the formula RCON— are acetylamino and benzoylamino.

A preferred group of the formula RN═N— is phenylazo.

A preferred group of the formula RS— is ethylthio.

A preferred group of the formula RSO₂— is methylsulfonyl.

A preferred group of the formula RSO— is methylsulfinyl.

A preferred group of the formula RSO₂O— is methylsulfonyloxy.

A preferred group of the formula RSOO— is methylsulfinyloxy.

One particularly preferred CIM of this invention is the prototypalcompound, Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide, that is, acompound of formula (2), wherein X₁=X=allyl, m=1, and n=0.

Isomeric mixtures of the compounds of formula (2) can be synthesized bymethods well known in the art. See, e.g., U.S. Pat. Nos. 4,643,994 and4,665,088, Block et al., J. Am Chem. Soc., 108, 7045-7055 (1986) andSendl et al., Planta Med., 57, 361-362 (1991), the complete disclosuresof which are incorporated herein by reference. The isomeric mixtures canthen be separated as described below to obtain a desired CM such asZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.

In particular, U.S. Pat. No. 4,665,088 describes the synthesis of(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide. Briefly, garlic issubjected to any convenient extraction procedure which acts to isolatethe allyl disulfide oxide component of garlic so that this component canbe thereafter dissolved in an appropriate lower alkanol for a time andat a temperature sufficient to form(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide. To obtain higheryields, the garlic should be freshly cut, chopped or ground. Wholegarlic cloves reduce yield, but can be satisfactorily used. The garlicpieces are blended with a volatile, water-miscible organic solvent suchas a lower alkanol, ether, or acetone and are allowed to sit for severalhours or days. The particulate material is usually removed prior tofurther processing. Vacuum concentration of the liquid and extraction ofthe aqueous residue with an appropriate solvent, such as diethyl ether,appears to increase the yield significantly. The extracted aqueousresidue can be washed several times with water, dried and evaporated toincrease the purity of the oil allyl disulfide oxide residue. The oilyresidue product is then dissolved in a volatile organic solvent, such asacetone or a lower alkanol in mixture with water (10-90%), andmaintained at a temperature of from about −40° C. to a temperature lessthan about the reflux temperature of the organic solvent in mixture withthe water. Generally, the higher the temperature, the lower the amountof time the mixture must be maintained at that temperature. It isgenerally desirable to adjust temperature to achieve a maintenance timeof several-hours, usually from about 10-72 hours.(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide can also be prepared asdescribed in the next paragraph.

U.S. Pat. No. 4,643,994 describes the synthesis of isomeric mixtures ofseveral compounds coming within the scope of formula (2) and describes ageneral synthetic scheme that can be used for preparing isomericmixtures of compounds of formula (2). Briefly, an appropriate disulfidehaving the formula:X₁—S—S—Z   (3)wherein Z is —(CH₂)_(m)—CH═CH—(CH)_(n)—, m, n, and X₁ are defined above,is treated with an oxidizing agent, preferably in the presence of asolvent, and preferably at a temperature of from about −40° C. to about65° C. to produce a thiosulfinate of the formula:

The thiosulfinate is then heated, typically refluxed, in the presence ofan appropriate solvent, preferably a 60:40 organic solvent:water mixtureto form a trithio oxide of the formula:

Typically, the-reaction which causes the formation of the trithio oxideof formula (5) also causes the formation of minor products wherein eachX₁ or Z can be Z or X₁, respectively. If a mixture of disulfides orthiosulfinates are used as starting compounds, the product will be afurther mixture of products. The mixture of products can be separated atthis point in the process by various means, such as extraction, or themixture can be maintained as such through the next step(s). The compound(5) can be used directly to make CIM's of the invention or can befurther oxidized to produce additional compounds of formula (2).Treatment with a stoichiometric (or slight excess) amount of oxidizingagent forms compounds of formula:

Treatment with further oxidizing agents at −30° C. to 40° C. producescompounds of formula:

Continued treatment with an oxidizing agent produces compounds offormula:

Each of compounds (7) and (8) can be reacted with a thiol of the formulaX₁SH, or an alkali metal salt thereof, wherein X₁ is defined above, toproduce further compounds having different selected substitutents as theX₁ moiety. These compounds can be purified by various means, includingextraction, arid used to make CIMs.

The CIMs can be purified from an isomeric mixture by high performanceliquid chromatography (HPLC) on columns packed with sorbents that arecapable of separating individual enatiomers from mixtures thereof. Seethe Examples below.

By this invention, safe and effective doses of the CIMs may inhibit theprogression of an HIV infection in a patient or the infection of anuninfected patient by HIV. Specifically, Z(−)-ajoene is such aninhibitor, although other CIMs may also be used.

One of the characteristics the inhibition of HIV infection is thediminution of the formation of HIV-induced syncytia, in which HIV targetcells, such as lymphocytes and monocytes, fuse together to form giant,multinucleate cells in HIV-infected patients. Transfer of geneticmaterial between cells will, thereby, also be inhibited by CIMs, whichinhibit fusion with cell membranes.

Additionally, because CIMs modulate pertinent integrin activity, itappears that CIMs inhibit the entry of the infective HIV material intoits target cells, including CD4-negative cells, both virus-to-cell andcell-to-cell entry, and the production of HIV and other viruses by theinfected cells. For these purposes, CIMs are preferably administered ina sufficient dose to provide a concentration approaching or exceeding 5micromoles per liter of patient's blood plasma, although lesserconcentrations may also be effective. This dose is effective when theCIMs are used alone or in a bi- or multi-therapy addressing differentdisease parameters (e.g., the function of viral enzymes).

In addition to infections caused by HIV and other viruses of theRetroviridae family, CIMs can be used locally or systemically to inhibitor prevent the transmission, in vivo and in vitro, of other virusesinfecting humans and other animals. In particular, CIMs can be used totreat or prevent infections caused by viruses, the transmission of whichinvolves fusion of at least a part of the virus with the membrane of thetarget cell that is to be infected. Such viruses include all envelopedviruses and other viruses that infect cells in this manner. Theenveloped viruses include the Retroviridae, Herpesviridae (e.g., herpessimplex, HSV-2, varicella zoster, Epstein-Barr virus, and cytomegalyvirus), Hepadnaviridae (e.g., hepatitis B), Flaviviridae (e.g., yellowfever virus and hepatitis C virus), Togaviridae (e.g. rubivirus, such asrubella virus, and alphavirus), Orthomyxoviridae (e.g., influenzavirus), Paramyxoviridae (e.g., measles, parainfluenza, mumps and caninedistemper viruses), Poxviridae (e.g., variola virus and vaccinia virus),and Rhabdoviridae (e.g., rabies virus). Other viruses that infect cellsby fusing with the membrane include Papovaviridae (e.g.,papillomavirus), Picornaviridae (e.g., hepatitis A virus andpoliomyelitis virus), Rotaviridae, and Adenoviridae. In addition toinhibiting or preventing virus-to-cell entry, CIMs can also inhibit orprevent cell-to-cell transmission of viruses (e.g., by inhibiting orpreventing syncytia formation or by inhibiting or preventingintercellular viral transfer-between cells in contact or closeproximity) and the production of viruses by infected cells.

CIMs also serve as agents that inhibit the adhesion, migration (e.g.,chemotaxis or is other infiltration of the tissue), and aggregation ofvarious cell types and lines, including blood platelets and neutrophils.CIMs, therefore, exhibit benefit as agents for the treatment ofpathologies derived from adhesion, migration, and aggregation of theseand other cells, including thrombosis and various types of inflammation.

Thrombosis is defined as blockage of blood vessel(s) by thrombi, i.e.,clots formed from fibrin and platelet aggregates, deposited on the innersurface of the vessel. Thrombi form in arteries (e.g., damaged as aresult of a disease) or in veins (e.g., due to lengthy immobilization).If a thrombus or a blood clot is dislodged and moves through thebloodstream to create an obstruction outside the place of its formation,it becomes an embolus (hence the terms “thromboembolism” and“thromboembolic disease”). Thrombosis or thromboembolism of coronaryarteries can cause heart attacks and myocardial infarction; the sameprocesses in brain arteries cause stroke. Inhibition of plateletaggregation by CIMs would, therefore, arrest thrombosis at early stages,precluding the development of thrombotic and thromboembollic diseases.

Inflammation, a pathological process inherent in a variety of distinctdiseases and illnesses, is defensive in nature, but potentiallydangerous if uncontrolled. When viewed at the “whole body” level, aninflammation is most frequently characterized by several localized.manifestations (indices), including hemodynamic disorders (e.g.,hyperemia and edema), pain, temperature increment, and functionallesion. These inflammatory phenomena are underlain by events at thecellular and molecular levels. At the cellular level, inflammation ischaracterized by leukocyte extravasation (a process involving adhesionof leukocytes to the endothelium of the vessel wall and migration intotissue where they may phagocytose bacteria, viruses, and cell debris)and platelet aggregation (a mechanism, inter alia, whereby the spread ofthe infection is prevented). At the molecular level, inflammation ischaracterized by activation of at least three plasma defense systems(complement, kinin, and coagulation/fibrinolysis cascades) and bysynthesis of cytokines and eicosanoids. When inflammation becomesgeneralized (as in the case of shock, for example), various indices ofinflammation occur systemically throughout-the entire organ/organism.

In cases of shock, platelets and leukocytes (principally neutrophils)aggregate in the blood vessels, leading to the development of a clinicalcondition known as multiple organ failure. The primary organ affected inshock patients is commonly the lung. Lung failure, or adult respiratorydistress syndrome (ARDS), a destructive inflammation resulting fromadhesion, aggregation, and degranulation of activated neutrophils in thepulmonary microvasculature, may be the main cause of death in patientssuffering shock. CIMs may thus counteract at least part of the effectsof shock, whether arising, for example, from sepsis, anaphylaxis, bloodloss, or from other precipitating events.

CIMs can also be administered in effective dosages to suppress manyother acute inflammatory processes, such as those associated withperitonitis, meningitis, and ischemia-reperfusion. Ischemia-reperfusioninjury occurs (e.g., in heart, brain, kidney, liver, lung, intestinaltract, or any limb) when blood supply is abruptly stopped (ischemia) andthen resumed (reperfusion) after a short period.

With the onset of ischemia and the decrease in the perfusion pressure,neutrophils are retained in the capillaries. As the ischemia progresses,cytokines (and other chemoattractants) are released into the capillarylumina in regions of the tissue where the blood flow blockage hasoccurred, increasing the adhesiveness of the retained neutrophils to theendothelium and to each other. Aggregates of neutrophils thus formedobstruct postcapiliary venules (“no-reflow,” or “no-washout”) andattenuate the restoration of the blood flow in the affected region,precluding its reoxygenation and extending the area of ischemia,Activated neutrophils trapped in the capillaries also release hydrolyticenzymes and reactive oxygen species (i.e., the armamentarium ordinarilyused to defend the host against microorganisms), producing a destructiveinflammation.

Restoration of the blood flow, however, further augments the severity ofthe inflammation thus developed. Neutrophils arriving to the previouslyischemic region are activated (by chemoattractants and/or productsreleased by the trapped neutrophils) and recruited into the tissue,where the defensive machinery of the cells is once again used againstthe host (secondary injury). Ischemia-reperfusion injury can also begeneralized, e.g., in the case of resuscitation after hemorrhagic shock(Mazzone, A., et al., Leukocyte CD11/CD18 Integrins: Biological andClinical Relevance, 80 Haemtatologica 161, 1995; see also, Reinhart W.H., Hemorheology: Blood Flow Hematology, 125 Schweiz. Med. Wochenschr.387, 1995).

CIMs are also potent inhibitors of adhesive interactions for othercells, such as lymphoid cells. Adhesion of lymphocytes to each other andnonlymphoid cells is prerequisite: to the development of any immuneresponse. CIMs may, therefore, serve as agents for the prevention,treatment, and control of adverse, undesirable, and self-destructiveimmune responses.

One group of such immunopathologies comprises diseases stemming fromdivers allergic reactions (e.g., delayed type hypersensitivity, Arthusreaction, and anaphylaxis). Allergy is an anomalous immune response toantigen challenge, characterized by recruitment of specific leukocytesubsets (e.g., cytotoxic lymphocytes and/or eosinophils) to the tissue,resulting in inflammation. Development of allergic inflammation is themain component in the pathogenesis of many diseases and illnesses,including, e.g., asthma, eczema, purpura pigmentosa chronica, variousvasculitides, and hay fever, in addition to those mentioned above. CIMsserve to control these diseases and illnesses.

Allograft rejection is another example of an undesirable immuneresponse, in which the transplanted organ is recognized by the immunesystem as a foreign body (“non-self”) and attacked in sequence bycytotoxic lymphocytes and phagocytes recruited from the circulation.This inflammatory response results in progressive disruption of thetissue, including graft necrosis. CIMs may be used to prevent cellrecruitment into transplanted tissue and thereby prolong graft survivalby reducing both acute and chronic aspects of rejection.

Moreover, the transplanted organ also contains lymphocytes, which, inturn recognize their new environment as “non-self.” The immune responseinitiated by these donor lymphocytes in the body of the recipientproduces a condition known as graft-versus-host disease (GVHD), whichcan lead to injury, both acute and chronic. CIMs can contribute to thecontrol of both acute and chronic GVHD.

Any method of treatment that suppresses both rejection andsusceptibility to viruses (which, like cytomegaly virus, frequentlycontaminate the transplanted organs and decrease the probability oftheir engraftment) will have an extra benefit to the graft recipient. Asdiscussed above, CIMs of instant invention exert pronounced antiviraleffects, in addition to being potent anti-inflammatory agents. Thus,administration of CIMs to patients undergoing organ transplantationoffers much promise as a novel therapeutic approach to the prevention ofrejection.

Self-destructive responses are caused by the failure of the immunesystem to distinguish “self” from “non-self” This group ofimmunopathologies comprises a wide variety of diseases.(herein termedcollectively “autoimmune diseases”), including, without limitation,rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome,multiple sclerosis, insulin-dependent diabetes mellitus,glomerulonephritis, Graves, disease, Hashimoto's thyroiditis, andvasculitides. Other-conditions and diseases may also fall into thiscategory (see, e.g., the discussion of psoriasis below) or comprise acomponent that does so (chronic viral diseases stimulating an autoimmuneresponse). In spite of pronounced differences in the clinical picture ofthe various autoimmune diseases, the underlying mechanisms involve, inevery case, undesirable recruitment of leukocytes to organs/tissuesaffected, resulting in destructive inflammation. CIMs can be used toreduce or prevent this cellular recruitment and thereby suppress theabnormal immune response. Accordingly, CIMs may be used to treat autoimmune diseases.

The beneficial effects of CIMs are achieved because these substances aremodulating the activity of integrins. As used herein, “modulate” meansto affect the development or expression of modalities normallycharacterizing the ability of a particular integrin to perform any ofits functions. A modulating agent may act on an integrin directly, e.g.,by binding to or interacting with a portion of at least one subunit(alpha or beta) of the integrin. The agent may also act in some otherfashion that is not considered direct, e.g., through any of the variouscellular substances and structures which ordinarily interact with orenable the participation of specific integrins, alone, or incombination. These substances and structures include, withoutlimitation, transmembrane proteins (e.g., integrins themselves andintegrin-associated proteins), membrane phospholipids, intracellularmolecules with messenger-like function (e.g. integrin-modulatingfactor), enzymes, and regulatory and signaling proteins. Thus, forexample, a modulation may result from alteration in integrinconformation, disassociation of the alpha and beta integrin subunits (orany of the parts thereof), disassociation of integrin clusters (and ofclusters formed by integrins with other proteins), or from the loss orvariations of integrin-cytoskeleton connections, although modulation mayalso occur from other types of effects. The functions of integrins, asdefined herein, are interrelated and include, inter alias signaling,adhesion, fusion, and internalization.

As a result of its ability to modulate activities in which integrinsparticipate, CIMs can be used to treat a plurality of diseases orconditions that involve undesirable integrin-mediated functions as amechanism, including those described above. For instance, CIMs can beused to inhibit virus-cell fusion. Moreover, CIMs may be used to inhibitundesired cell-cell fusion.

Undesired cell-cell fusion can include, for example, cell-cell fusion(transitory or permanent) that results in the transfer of viral geneticmaterial; cell-cell fusion that results in the formation ofmultinucleate cells (e.g., syncytia, giant cells, and osteoclasts);undesired fertilization of eggs by sperm; and the formation ofmultinucleate germinal cells (syncytiotrophoblast).

Thus, CIMs can be used as a contraceptive, being administered pervaginam (topically), per os, or in any other appropriate way, when usedfor this purpose. CIMs can prevent conception, however, at the stage ofembryo implantation. For example, CIMs can prevent the initial adhesionof the blastocyst to the endometrium and the migration ofcytotrophoblasts through the maternal epithelium (i.e., processessimilar to certain steps in the leukocyte extravasation cascade andtumor cell metastasis).

Furthermore, CIMs are capable of inhibiting cytotrophoblast invasion, aprocess differing from extravasation in that it goes from the tissues tothe vascular lumen and that the invading cells cross the blood-tissuebarrier from outside of the vessel. For example, the production ofproteolytic enzymes that are used by cytotrophoblasts to penetrate thebasement membrane is governed by integrin outside-in signaling.Modulation of the signaling function of integrins by CIMs can eithercompletely prevent the production of the requisite enzymes or attenuateit to an extent precluding-invasion. A related mechanism underlies theability of CIMs to block angiogenesis, preventing the blood supply tothe fetal tissue. Thus, CIMs can be used as effective emergencycontraceptives to prevent unwanted pregnancy or interrupt it at an earlystage.

There are yet other mechanisms whereby CIMs, when desired, can exertcontraceptive effects. For example, they can prevent the chemotacticresponse of sperm in the vaginal environment (a specific case of cellhoming) and sperm-interactions with the epithelium of the female genitaltract. Moreover, CIMs can be administered to males to modulate integrinsin sperm precursors and other testicular or epididymal cells, therebyinterfering with the maturation processes and resulting in theproduction of fertilization-incompetent gametes or inhibition offertilization-competent gametes.

The development of major bone diseases, including osteoporosis, isunderlain by excessive bone resorption. This fundamental function isperformed by osteoclasts. Osteoclasts are unique multinucleate bonecells formed by fusion of mononuclear progenitors called preosteoclasts.The regulation of osteoclast formation may be achieved by agents actingat various levels of osteoclast formation, including preosteoclastfusion (Zaidi, et al., Cellular Biology of Bone Resorption, 68 Biol.Rev. 197, 1993). CIMs can regulate bone resorption because it caninhibit the fusion of preosteoclasts-necessary for the formation ofosteoclasts.

Granulomas are characteristic of chronic inflammatory lesions, such asthose found, in tuberculosis and other chronic infections. Granulomasare also present in sarcoidosis, a chronic, systemic inflammatorydisease of undescribed etiology. Granulomas present in cases of chronicinfection and in sarcoidosis contain a large number of multinucleategiant cells formed by the fusion of macrophages. Other diseasesassociated with the formation of multinucleate cells include, withoutlimitation, Crohn's disease, Langerhans cell histiocytosis; and giantcell arteritis. CIMs can be used to inhibit the formation of these giantmultinucleate cells with beneficial therapeutic effects.

Excessive formation of fibrous interstitial tissue (i.e., fibrosis, orsclerosis) is characteristic of certain diseases (such as sclerodermaand idiopathic pulmonary fibrosis) and an outcome of chronicinflammatory processes (e.g., glomerular fibrosis). The development offibrotic lesions and progression of fibrosis, associated with theseconditions, diseases, and illnesses, has been linked to abnormalintegrin expression and altered cell adhesion patterns. CIMs can,therefore, be used for treatment of fibrotic lesions, including, theformation of keloid (scar tissue).

Lesions observed in skin diseases and illnesses of diverse origin, suchas lichen planus, urticaria, dermatofibroma, psoriasiform dermatitides,and keratoses, are characterized by aberrant integrin expression. CIMscan serve as an agent for the symptomatic treatment of these diseases,being administered topically, intradermally, and subcutaneously at thesite of lesions, or in any other appropriate way, when used for thispurpose.

Another disease characterized by the formation of cutaneous lesions ispsoriasis. Although the etiology of psoriasis attends furtherelucidation (several viruses and an autoimmune component could beinvolved), its pathogenesis is associated with abnormal expression ofintegrins in target tissue (e.g., in vascular cells, keratinocytes, anddendritic cells), proliferation of endothelial and epidermal cells, andan autoimmune component (recruitment of lymphocytes and macrophages toskin and joints). CIMs, therefore, can be used to treat psoriasis inmultiple respects.

As a result of their antiviral and anti-inflammatory activity, CIMs arelikely to exhibit significant potency in the prevention and treatment ofcertain diseases with combined etiopathogenesis. As roughly elaboratedherein, the term “etiopathogenesis” is used in reference to diseases forwhich no distinction can be drawn thus far as to the etiology andpathogenesis. In addition to psoriasis, mentioned above, a good exampleof such a disease is atherosclerosis. A variety of viruses mayparticipate in the development of atherosclerosis. One of the beststudied viral contributors is cytomegalovirus, which induces a specifictype of infection characterized by plaque formation along the bloodvessels (Melnick, J. L., et al., Cytomegalovirus and Atherosclerosis, 17BioEssays 899, 1995). A prominent feature of atherosclerosis is therecruitment of monocyte-macrophages into atherosclerotic plaques, whichis an integrin-dependent process. Also, proliferation of smooth musclecells, which contributes to the formation of atheroscierotic lesions, isregulated by integrins. As indicated elsewhere in this document, CIMsinhibit the transmission of viral infections virus-to-cell-andcell-to-cell. Integrin-mediated adhesion and signaling are alsoinhibited by CIMs. Thus, CIMs, for multiple reasons, can be used totreat diseases involving combinations of integrin-dependentetiopathogenetic factors, including those that are in part of viraletiology.

Certain neurodegenerative-disorders of unclear etiology (e.g.,Alzheimer's disease and amyotrophic lateral sclerosis) involveautoimmune inflammation of nervous tissue as a pathogenetic mechanism.CIMs should, therefore, demonstrate'significant potency in mitigatingthe symptoms of these diseases and slowing their progression. Thisconclusion is further supported by various studies which show that otheranti-inflammatory treatments benefit Alzheimer's patients (McGeer, P.L., et al., The Inflammatory Response System of Brain: Implications forTherapy of Alzheimer's and Other Neurodegenerative Diseases, 21 BrainRes. Brain Res. Rev. 195, 1995; Breitner, J. C., et al., Delayed Onsetof Alzheimer's Disease with Nonsteroidal Anti-Inflammatory and HistamineH2 Blocking Drugs, 16 Neurobiol. Aging 523, 1995).

To treat or prevent any of these disorders, diseases or conditions, aneffective amount of a CIM is administered to an animal in need thereof.Preferably, the animal is a mamnmal, such as a rabbit, goat, dog, cat,horse or human. Effective dosage forms, modes of administration, anddosage amounts may be determined empirically, and making suchdeterminations is within the skill of the art. It is understood by thoseskilled in the art that the dosage amount will varywith the disorder,disease orcondition to be treated or prevented, the severity of thedisorder, disease or other condition, which integrin(s) is (are) to bemodulated, the route of administration, the rate of excretion, theduration of the treatment, the identity of any other drugs beingadministered, the age, size and species of animal, and like factors wellknown in the arts of medicine and veterinary medicine. In general, asuitable daily dose of a CRA will be that amount of the compound whichis the lowest dose effective to produce the desired effect. Theeffective daily dose of a CIM maybe administered as two, three, four,five, six or more sub-doses, administered separately at appropriateintervals throughout the day. An existing disorder, disease or conditiontreated with a CIM or combination of CIMs according to the invention maybe reduced, inhibited, suppressed or eliminated or one or more symptomsof the disorder, disease or condition may be alleviated or eliminated.

CIMs may be administered in any desired and effective manner: aspharmaceutical compositions for oral ingestion, or for parenteral orother administration in any appropriate manner such as intraperitoneal,subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal,vaginal, sublingual, intramuscular, intravenous, intraarterial,intrathecal, or intralymphatic. For instance, the topical application ofCIMs to mucous membranes (in the form of creams, gels, suppositories,and other known means of topical administration) can be used to preventHIV infection of mucosal cells, an important route of HIV transmission.In addition, intralymphatic administration of CIMs maybe advantageous inpreventing the spread of HIV within the body. Further, CIMS may beadministered in conjunction with other treatments for the disorder,disease or condition being treated with the CIM, such as other antiviraldrugs, other contraceptives, and other anti-shock or anti-inflammatorydrugs or treatments. CIMs maybe encapsulated or otherwise protectedagainst gastric or other secretions, if desired.

While it is possible for a CIM of the invention to be administeredalone, it is preferable to administer the CIM as a pharmaceuticalformulation (composition). The pharmaceutical compositions of theinvention comprise one or more CIMs as an active ingredient in admixturewith one or more pharmaceutically-acceptable carriers and, optionally,one or more other compounds, drugs, ingredients and/or materials.Regardless of the route of administration selected, the CIMs of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton,Pa.).

Pharmaceutical carriers are well known, in the art (see, e.g.,Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.)and The National Formulary (American Pharmaceutical Association,Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol,and sorbitol), starches, cellulose preparations, calcium phosphates(e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogenphosphate), sodium citrate, water, aqueous solutions (e.g., saline,sodium chloride injection, Ringer's injection, dextrose injection,dextrose and sodium chloride injection, lactated Ringer's injection),alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol),polyols (e.g., glycerol, propylene glycol, and polyethylene glycol),organic esters (e.g., ethyl oleate and tryglycerides), biodegradablepolymers (e.g., polylactide-polyglycolide, poly(orthoesteis), andpoly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils(e.g., corn germ, olive, castor, sesame, cottonseed, and groundnut),cocoa butter, waxes (e.g. suppository waxes), paraffins, silicones,talc, silicylate, etc. Each carrier used in a pharmaceutical compositionof the invention must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to theanimal. Carriers suitable for a selected dosage form and intended routeof administration are well known in the art, and acceptable carriers fora chosen CIM, dosage form and method of administration can be determinedusing ordinary skill in the art.

The pharmaceutical compositions of the invention may, optionally,contain additional ingredients and/or materials commonly used inpharmaceutical compositions. These ingredients and materials are wellknown in the art and include (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, suchas carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, suchas glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,sodium starchlglycolate, cross-linked sodium carboxymethyl cellulose andsodium carbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and glycerol monosterate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,and sodium lauryl sulfate; (10) suspending agents, such as ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth; (11) buffering agents; (12) excipients, such as lactose,milk sugars, polyethylene glycols, animal and vegetable fats, oils,waxes, paraffins, cocoa butter, starches, tragacanth, cellulosederivatives, polyethylene glycol, silicones, bentonites, silicic acid,talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, andpolyamide powder; (13) inert diluents, such as water or other solvents;(14) preservatives; (15) surface-active agents; (16) dispersing agents;(17) control-release or absorption-delaying agents, such ashydroxypropylmethyl cellulose, other polymer matrices, biodegradablepolymers, liposomes, microspheres, aluminum monosterate, gelatin, andwaxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21)emulsifying and suspending agents; (22), solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan; (23)propellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane; (24) antioxidants; (25) agentswhich render the formulation isotonic with the blood of the intendedrecipient, such as sugars and sodium chloride; (26) thickening agents;(27) coating materials, such as lecithin; and (28) sweetening,flavoring, coloring, perfuming and preservative agents. Each suchingredient or material must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the animal. Ingredients and materials suitable for aselected dosage form and intended route of administration are well knownin the art, and acceptable ingredients and materials for a chosen CIM,dosage form and method of administration can be determined usingordinary skill in the art.

Pharmaceutical formulations suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules, asolution or a suspension in an aqueous or non-aqueous liquid, anoil-in-water or water-in-oil liquid emulsion, an elixir or syrup, apastille, a bolus, an electuary or a paste. These formulation can beprepared by methods known in the art, e.g., by means of conventionalpan-coating, mixing, granulation or lyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared by mixing theactive ingredient(s) with one or more pharmaceutically-acceptablecarriers and, optionally, one or more fillers, extenders, binders,humectants, disintegrating agents, solution retarding agents, absorptionaccelerators, wetting agents, absorbents, lubricants, and/or coloringagents. Solid compositions of a similar type maybe employed as fillersin soft and hard-filled gelatin capsules using a suitable excipient. Atablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared using asuitable binder, lubricant, inert diluent, preservative, disintegrant,surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine. The tablets, and other solid dosageforms, such as dragees, capsules, pills and granules, may optionally bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.They may also be formulated so as to provide slow or controlled releaseof the active ingredient therein. They may be sterilized by, forexample, filtration through a bacteria-retaining filter. Thesecompositions may also optionally contain opacifying agents and may be ofa composition such that they release the active ingredient only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more activeingredient(s) with one or more suitable nonirritating carriers which aresolid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound. Formulations which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such pharmaceutically-acceptablecarriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, drops and inhalants. The active compound may be mixed understerile conditions with a suitable pharmaceutically-acceptable carrier.The ointments, pastes, creams and gels may contain excipients. Powdersand sprays may contain excipients and propellants.

Pharmaceutical compositions suitable for parenteral administrationscomprise one or more CIMs in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain suitable antioxidants,buffers, solutes which render the formulation isotonic with the blood ofthe intended recipient; or suspending or thickening agents. Properfluidity can be maintained, for example, by the use of coatingmaterials, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. These compositions mayalso contain suitable adjuvants, such as wetting agents, emulsifyingagents and dispersing agents. It may also be desirable to includeisotonic agents. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption.

In some cases, in order to prolong the effect of a drug, it is desirableto slow its absorption from subcutaneous or intramuscular injection.This maybe accomplished by the use of a liquid suspension of crystallineor amorphous material having poor water solubility. The rate ofabsorption of the drug then depends upon its rate of dissolution which,in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugmay be accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms may be made by forming microencapsulematrices of the active ingredient in biodegradable polymers. Dependingon the ratio of the active ingredient to polymer, and the nature of theparticular polymer employed, the rate of active ingredient release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and-may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

The invention further provides a method of modulating anintegrin-mediated function of one or more cells. The method comprisescontacting the cell(s) with an amount of a CM of the invention effectiveto modulate the integrin-mediated function. Methods of contacting cellsin vivo are the same as those described above for treating a disorder,disease or condition. Methods of contacting cells in vitro withcompounds (e.g., placed in a solution, such as a cell culture medium,containing the compound) are well known in the art. Suitable conditions(time, temperature, concentrations, type of medium, etc.) are known orcan be determined empircally as is well known in the art.

The invention also provides a method of treating a tissue by contactingthe tissue with a CIM. Such treatment improves the condition of thetissue for subsequent use, as compared to tissue which is not treatedwith a CIM. In particular, tissue which is to be transplanted into arecipient may be treated with a CIM, preferably before excision, or, ifnot, at the time of excision, and the chances of the tissue beingsuccessfully transplanted will be increased.

The tissue to be treated may be any tissue. For instance, the tissue maybe an organ (such as a heart, blood vessel, lung, liver, kidney, ski,cornea, or part of an organ, such as a heart valve), or a non-organtissue (such as bone marrow, stem cells, or gametes). The tissue istreated by contacting it one or more times with an effective amount of aCIM. Methods of contacting tissues with agents are well known in theart. For instance, the contacting can be accomplished conveniently byrinsing or perfusing the tissue with, and/or submersing the tissue in, asolution of the CIM in a physiologically acceptable diluent. Contactingcan also include perfusing the tissue or the donor of the tissue (e.g.,a brain-dead human) with a solution of the CIM in a physiologicallyacceptable diluent prior to excision of the tissue.Physiologically-acceptable diluents are those that are compatible with,and not harmful to, the CIM and the tissue. Such diluents are well knownand include saline and other solutions and fluids.

Effective amounts of the CIM may be determined empirically, and makingsuch determinations is within the skill of the art. It is understood bythose skilled in the art that the amount may vary as a result of one ormore factors, including the type and size of the tissue, the intendeduse of the tissue, the length of storage of the tissue before use, theidentity of any other agents being used, the number of treatments, andlike factors well known in the art.

The CIM agent may be used in conjunction with other agents to treattissue. For instance,.the tissue may also be treated with preservationagents (i.e., agents which inhibit deterioration of the condition of thetissue), antibiotics, antifungal drugs, antiviral drugs,antiinflammatory drugs, or other treatments (e.g., lung surfactants inthe case of lung tissue).

After being contacted with the CIM, the tissue may be used immediatelyor may be stored until needed. Methods of storing tissue are well knownin the art. The tissue may be stored in contact with the CIM. Tissuesare preferably stored at low temperatures, typically 4-18° C., andnon-organ tissues may typically be frozen. The time of storage willvary, depending on the type of tissue, the storage environment(including the temperature of storage), and the intended use. Such timescan be determined empirically, and making such determinations is withinthe skill in the art. Regardless of the length and conditions ofstorage, timely treatment with a CIM mitigates the effects of harvestand/or storage, and treated tissues will be in better condition thantissues not treated with a CIM.

Tissues treated with CIMs may be used for a variety of purposes. Forinstance, they may be transplanted into recipients. They may also beused for research purposes; such as studying the function of the tissue.

It is taken that CIMs may induce a rest state in tissue, which lastsuntil the CIMs are removed. In addition, treatment of a tissue with anCIM improves the condition of the tissue by reducing the negativeeffects and consequences of harvesting and storing tissue. For instance,CIMs inhibit (prevent or reduce) the adhesion and aggregation of cellswhich would otherwise cause injury to a tissue (seethe discussionabove). Thus, treatment of a tissue with a CIM prevents or reducesdamage to the tissue.

In particular, ischemia (anemia due to constriction or obstruction of ablood vessel) occurs upon harvesting an organ. Both the injury due toischemia and that due to reperfusion after ischemia (which generallyoccurs upon resuming blood flow in an organ, such that occurring whentransplanting an organ into a recipient), can be inhibited by treatmentof the organ with a CIM. To achieve maximum inhibition of ischemicinjury and ischemic reperfusion injury, the organ should be contactedwith the CIM before or immediately after harvesting of or interruptionof normal blood supply to the organ, to mitigate the rapid onset ofinjury and other changes associated with ischemic injury. Such treatmenthas beneficial effects even for organs that are to be used immediately,such as in the case of many transplants. Preferably the contacting takesplace by perfusing the organ with a solution comprising the CIM.

For organs that are stored (even for a short time), benefits areobtained by contacting the organ with a CIM immediately prior to use.This treatment may serve, among other purposes, to eliminate the effectsof any cytokines that may have been produced, as well as to preventadhesion and aggregation of cells which would otherwise cause tissueinjury. The treatment of an organ after storage can be the firsttreatment of the organ with a CIM, or can be the second treatment of anorgan which was first treated before or immediately after harvest.Again, the organ is preferably treated by perfusion with a solutioncomprising the CIM.

As discussed above, CIMs suppress undesired immune responses. Thus,treatment of a tissue with a CIM prior to transplantation acts as aninitial treatment for the prevention of graft rejection and/or graftversus host disease (GVHD) in transplant recipients.

Of course, the recipient may receive additional amounts of an CIM toprevent graft rejection and/or GVHD as described above. The amountadministered to the recipient should also be chosen so that inhibitionof injury due to ischemia and ischemia reperfusion is continued.

Finally, CIMs can inhibit the transmission of viral infections from atissue to a recipient of transplanted tissue and vice versa (see thediscussion above). This includes all of the viral infections describedabove.

The invention also provides a kit for treating tissues. The kit is apackaged combination of one or more containers holding reagents andother items useful for treating tissues according to invention. The kitcomprises a container holding a CIM. Suitable containers includebottles, bags, vials, test tubes, syringes, and other containers knownin the at. The kit may also contain other items which are known in theart or which may be desirable from a commercial and user standpoint,such as instructions for treating a tissue, a container for the tissue,diluents, preservation agents, antibiotics, antifungal drugs, antiviraldrugs, anti-inflammatory drugs, surfactants, buffers, empty syringes,tubing, gauze, pads, disinfectant solution, etc.

It should be understood that, whereas some integrin functions aremodulated by CIMs of formula (2), there maybe certain integrinfunctions, the modulation of which is achieved by the other three ajoeneenantiomers of instant invention and derivatives thereof. Thus, allintegrin-modulating substances incorporating the corresponding trithiaoxide chiral structures should be properly referred to as CIMs.

EXAMPLES Example 1 Methods of Ajoene Separation into Four Enantiomers

A 3:1 mixture of racemic Z- and E-ajoenes (unseparated ajoene) wasdissolved in heptane/ethanol/diethylamine (90:10:0.1) and separated byHPLC on a 4.6×250 mm Chiralpak AS column (Chiral Technlogies, Exton,Pa.) using a mobile phase of heptane/ethanol/diethylamine 90:10:0.1(flow rate, 1 mL/min; temperature, 25° C.). The detector recorded twoparameters: the absorption of the eluate at 254 nm and the rotation ofthe plane of polarization of polarized light passing through the eluate;in the latter case, upward and downward peaks corresponded,respectively, to dextrorotatory (+) and levorotatory (−) enantiomers.The resulting chromatogram contained eight peaks superimposed in such away that four pairs were clearly seen. In each pair, the two coincidingpeaks correspond to a particular enantiomer of ajoene, i.e., Z(+), Z(−),E(+), and E(−). The Z(+), E(+), E(−), and Z(−) enantiomers of ajoenewere eluted with retention times of 21.4, 23.8, 26.2, and 33.1 minutes,respectively.

In another experiment, the separation was achieved in the same system,the only difference being in the composition of the mobile phase(hexane/ethanol 90:10). Again, the Z(+), E(+), E(−), and Z(−)enantiomers were clearly separated (the respective elution times were20.4, 23.1, 24.6, and 31.2 minutes). The peaks were collected into taredpolypropylene tubes and stored on ice for 48 hours, after whichanalytical runs were performed with each fraction. The results of theanalysis demonstrated that very good separation of the isomers had beenachieved (˜98%) and that the isomers did not undergo racemization underthe conditions of the storage. Thereafter, the fractions were dried downby rotary evaporation (6 to 10 min at 35° C.) and re-analyzed under thesame conditions. The peaks present in the fractions showed theiroriginal retention times and no other peaks were present. Thus, thedrying down process did not cause racemization.

In a yet another experiment, HPLC separation of the enantiomers involveda different column (10×250 mm Chiralpak AD). The conditions used were asfollows: mobile phase, hexane/ethanol 90:10; flow rate, 6 mL/min;temperature, ambient; detector wavelength, 254 nm. Typically, 10 mg ofthe 3:1 mixture was loaded per run and fractions collected manually. Thefractions were dried down by rotary evaporation at 37° C., resulting inlight oil. The oil was taken up in anhydrous ether and dried down byrotary evaporation. The resulting light oils were stored at −80° C. insealed containers. Analytical runs on the same column demonstrated thatZ(+), E(+), E(−), and Z(−) enantiomers of ajoene had retention times of53 min, 58 min, 62 min, and 75 min.

Example 2 Inhibition of VLA-4-mediated Cell Adhesion A Method ofAssessment of Integrin-modulating Activity of the Four Enantiomers ofAjoene

Integrin-modulating activity of the enantiomers was compared in awell-defined system of inhibition of VLA-4-mediated adhesion ofenzyme-labeled PM1 cells (NIH AIDS Repository, Rockville, Md.) toVCAM-1-coated artificial substrata. In this experiment, enzyme-linkedimmunosorbent assay (ELISA) plates were coated with rabbit anti-humanIgG (Fc-specific). Aliquots of 100 μL of supernatant fromVCAM/IgG-secreting COS7 cells were added to each well, the platesincubated at 37° C. for 1 hour, and the wells washed withphosphate-buffered saline (PBS). Thereafter, 50 μL aliquots of theenantiomers (various dilutions in RPMI 1640 medium) were introduced toeach well, followed by addition of 50 μL PM1 cells (4×10⁶ per mL)labeled with horseradish peroxidase (HRP) by pinocytosis. The plateswere incubated at room temperature for 10 min, centrifuged (1000 rpm, 1min) and incubated once again at 37° C. for additional 10 min. Cellsthat failed to form VLA-4-dependent adhesive contacts with thesubstratum were washed off with PBS in two turns. Adherent cells werelyzed by adding to each well a buffered solution of the substrate,supplemented with 1% TritonX-100. The reaction was stopped with 0.5 MH₂SO₄, andthe optical density of the wells, read at 450 nm. The value ofthis parameter, characterizing the activity of the enzyme and the numberof the adherent cells, is inversely proportional to theintegrin-modulating activity of the compound.

The Z(−) enantiomer of ajoene consistently exhibited a 4-fold higheradhesion-inhibiting activity than any other enantiomer of the original3:1 mixture of racemic Z- and E-ajoenes.

Example 3 Differential Inhibition by Ajoene Enantiomers ofIntegrin-dependent Fusion Leading to Syncytium Formation in HIV-infectedCells

The purified enantiomers were taken up in DMSO at a concentration of 10mg/mL. Dilutions were made in RPMI 1640 medium containing 10% fetal calfserum and 10 mM HEPES (cRPMI). H9 cells (ATCC, Rockville, Md.) infectedwith HIV-1_(RF), (NIH AIDS Repository, Rockville, Md.) and uninfected H9cells were washed with cRPMI and resuspended in cRPMI at a density of4×10⁶ per mL. Uninfected cells (50 μL) were mixed with serial dilutionsof the compounds (100 μL) and incubated at 37° C. for 30 min beforeadding and mixing 50 μL of infected H9 cells. The plates were incubatedat 37° C. for 6 to 15 hours before scoring syncytium formation.

The results are presented in FIG. 1. As shown in FIG. 1, the Z(−)enantiomer was at least four times more active than its Z(+)counterpart, racemic E-ajoene, or the unseparated 3:1 mixture of racemicZ- and E-ajoenes (the respective values of IC₁₀₀ were 12.5 and 50micromoles per liter). A similar result was obtained in another system,where syncytium formation was induced by mixing and incubating togetherfor 13 hours 50 μl MT2 cells (NIH AIDS Repository) and 50 μl U937 cells(ATCC) infected with HIV-1_(RF)(both cell populations had the density of2×10⁶ per mL).

Taken together, Examples 2 and 3 demonstrate thatZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide is a stereoselective,chiral integrin modulator, the activity of which is significantly higherthan that of the parent racemates (E-, Z-ajoenes, and various mixturesthereof).

1. A pharmaceutical composition comprising a pharmaceutically-acceptablecarrier and Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 2. A methodof modulating an integrin-mediated function of one or more cellscomprising contacting the cell(s) with an amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide effective to modulate theintegrin-mediated function.
 3. The method of claim 2 wherein thefunction comprises adhesion, migration, aggregation, signaling, fusion,internalization, or combinations thereof.
 4. The method of claim 3wherein the aggregation of platelets is inhibited.
 5. The method ofclaim 3 wherein the migration, adhesion, or aggregation of neutrophilsis inhibited.
 6. A method of treating or preventing a disorder, diseaseor condition in which one or more integrins play a role comprisingadministering to an animal in need thereof an effective amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 7. A method of treatinga thrombotic disorder or a disease or condition arising therefromcomprising administering to an animal in need thereof an effectiveamount of Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 8. The methodof claim 7 wherein the disease or condition comprises ischemia,myocardial infarction or stroke.
 9. A method of treating inflammation oran inflammatory disease comprising administering to an animal in needthereof an effective amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 10. The method of claim9 wherein the inflammation is associated with peritonitis, meningitis,reperfusion after ischemia, delayed type hypersensitivity, an Arthusreaction, anaphylaxis, allograft rejection, graft-versus-host disease orarthritis.
 11. A method of treating or inhibiting the transmission of aviral infection comprising administering to an animal in need thereof aneffective amount of Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide. 12.The method of claim 11 wherein the viral infection is caused by a virus,the transmission of which involves fusion of at least a part of thevirus with the membrane of the cell that is to be infected, and theZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide inhibits the fusion ofthe virus with the cell membrane.
 13. The method of claim 12 wherein theviral infection is caused by a virus which is an enveloped virus. 14.The method of claim 13 wherein the viral infection is caused by a virusof the Retroviridae family, Hepadnaviridae family, Orthomyxoviridaefamily, Flaviviridae family, Togaviridae family, Paramyxoviridae family,Rhabdoviridae family, Poxviridae family or Herpesviridae family.
 15. Themethod of claim 14 wherein the viral infection is caused by a virus ofthe Retroviridae family.
 16. The method of claim 15 wherein theRetroviridae is HIV.
 17. The method of claim 12 wherein the viralinfection is caused by a virus of the Adenoviridae family,Picornaviridae family, Rotaviridae family or the Papovaviridae family.18. A method of treating arthritis comprising administering to an animalin need thereof an effective amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 19. A method of treatingor suppressing an adverse, undesirable or self-destructive immuneresponse comprising administering to an animal in need thereof aneffective amount of Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide. 20.The method of claim 19 wherein the immune response comprises an allergicreaction.
 21. The method of claim 20 wherein the allergic reaction isdelayed type hypersensitivity, an Arthus reaction or anaphylaxis. 22.The method of claim 19 wherein the immune response is allograftrejection or graft-versus-host disease.
 23. A method of treating anautoimmune disease comprising administering to an animal in need thereofan effective amount of Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.24. The method of claim 23 wherein the autoimmune disease is rheumatoidarthritis, systemic lupus erythematosus, Sjogren's syndrome, multiplesclerosis, insulin-dependent diabetes mellitus, glomerulonephritis,Graves disease, Hashimoto's thyroiditis, or vasculitides.
 25. A methodof treating psoriasis comprising administering to an animal in needthereof an effective amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 26. A method of treatingartherosclerosis comprising administering to an animal in need thereofan effective amount of Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.27. A method of treating a disorder, disease or condition involving aplurality of integrin-dependent mechanisms, wherein the disorder,disease, or condition is selected from the group consisting of athrombotic disease or disorder, shock, inflammation, inflammatorydisease, viral infection, arthritis, autoimmune disease, psoriasis,atherosclerosis, tumor metastasis, fertilization, cell-cell fusion,lesion formation, transfer of genetic information, and combinationsthereof, the method comprising administering to an animal in needthereof an effective amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 28. A method ofinhibiting metastasis of tumors, or inhibiting integrin-mediatedcarcinogenesis comprising administering to an animal in need thereof aneffective amount of Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide. 29.A method of contraception comprising administering to an animal in needthereof an effective amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 30. A method ofinhibiting undesirable integrin-mediated cell-cell fusion comprisingadministering to an animal in need thereof an effective amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 31. The method of claim30 wherein the undesired cell-cell fusion is cell-cell fusion thatresults in the formation of multinucleate cells or multinucleategerminal cells.
 32. The method of claim 31 wherein the multinucleatecells are syncytia, giant cells, or osteoclasts.
 33. A method ofinhibiting the formation of a lesion comprising administering to ananimal in need thereof an effective amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 34. The method of claim33 wherein the lesion is a granuloma.
 35. The method of claim 33 whereinthe lesion is a fibrotic lesion.
 36. A method of inhibiting the transferof genetic material comprising administering to an animal in needthereof an effective amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 37. The method of claim36 wherein the genetic material is viral genetic material.
 38. A methodof treating a tissue in order to inhibit degradation thereof comprisingcontacting the tissue with an effective amount ofZ(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.
 39. The method of claim38 wherein the tissue is excised from a donor and subsequentlytransplanted into a recipient.
 40. The method of claim 39 wherein thetissue is treated with the compound before excision, at the time ofexcision, after excision, or combinations thereof.
 41. The method ofclaim 39 wherein the tissue is stored in contact with the compound. 42.The method of claim 38 wherein the tissue is an organ.
 43. The method ofclaim 42 wherein the organ is a heart, a blood vessel, a lung, a liver,a kidney, skin, or a cornea.
 44. The method of claim 42 wherein theorgan is perfused with a solution comprising the compound.
 45. Themethod of claim 38 wherein the tissue is part of an organ.
 46. Themethod of claim 45 wherein the part of an organ is a heart valve. 47.The method of claim 38 wherein the tissue is a non-organ tissue.
 48. Themethod of claim 47 wherein the non-organ tissue is bone marrow, stemcells, or gametes.
 49. The method of claim 39 wherein an effectiveamount of the compound is administered to the recipient of the tissue atthe time of receiving the tissue, at one or more times after receivingthe tissue or both.
 50. A kit for treating a tissue comprising acontainer holding Z(−)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide.